Subcellular protein turnover in human neural progenitor cells revealed by correlative electron microscopy and nanoscale secondary ion mass spectrometry imaging

Protein turnover is a critical process for accurate cellular function, in which damaged proteins in the cells are gradually replaced with newly synthesized ones. Many previous studies on cellular protein turnover have used stable isotopic labelling by amino acids in cell culture (SILAC), followed by proteomic bulk analysis. However, this approach does not take into account the heterogeneity observed at the single-cell and subcellular levels. To address this, we investigated the protein turnover of neural progenitor cells at the subcellular resolution, using correlative TEM and NanoSIMS imaging, relying on a pulse-chase analysis of isotopically-labelled protein precusors. Cellular protein turnover was found significantly heterogenous across individual organelles, which indicates a possible relation between protein turnover and subcellular activity. In addition, different isotopically-labelled amino acids provided different turnover patterns, in spite of all being protein precursors, suggesting that they undergo distinct protein synthesis and metabolic pathways at the subcellular level.


Introduction
Protein turnover, the replacement of old or faulty proteins with newly synthesized ones, is a tightly regulated cellular process aimed at maintaining an intact proteome and ensuring proper functioning of cells.2][3] Protein turnover is dependent on transcription, translation, protein modications and degradation and it has been shown to differ between different cell types 4 and be inuenced by the extracellular environment. 5rotein turnover is also a reection of protein lifetime.The lifetimes of specic proteins have been determined in different mouse brain regions and the average lifetime was found to be around 10 days (most proteins had a lifetime from 3-13 days). 6NA binding, signalling, and RNA binding proteins were found to have shorter lifetimes whereas histones, myelin and extracellular matrix proteins were found to be long lived.Synaptic long-lived proteins have been found in rodents in in vitro neuronal cultures and in vivo, and their lifetime was found to change when the activity of neurons was modulated pharmacologically. 7Nevertheless, the majority of synaptic proteins was found to have a shorter lifetime of 2-5 days in cultured rat cortical neurons. 8hile providing valuable insight into the lifetimes of specic proteins, these data cannot provide insight into whether the protein's localization inuences its lifetime.Youse et al. have shown with stimulated emission depletion (STED) microscopy that the localization of specic synaptic proteins correlates to their lifetimes. 9In addition, the protein turnover of differently active synapses in cultured hippocampal neurons was found to be highly heterogenous, 10 highlighting the need to study the subcellular spatial organization of protein turnover, especially in highly polarized cells such as neurons and their precursors.
1][12][13][14][15] NanoSIMS has been used to study protein turnover in cells and tissues at a subcellular level for example in stress granules, 16 stereocilia, 17 synapses, 10 and lysosomes. 18In NanoSIMS, a primary ion beam erodes a sample surface pixel by pixel, generating atomic and diatomic secondary ions from the sample surface.The ions of a selected polarity are then transferred into a magnetic mass analyser and further detected in parallel by up to 7 mass detectors.The results are ion images showing the spatial distribution of these detected ions across the sample surface.NanoSIMS offers high sensitivity of detection (detection limit of ppb-ppm), high mass resolution (m/Dm ∼10 000), and especially, high spatial resolution (down to ∼50 nm), 19 which makes it well-suited for studying the chemical composition at a subcellular level.NanoSIMS has been used with rare isotopic labelling to track protein turnover in cells and tissues. 16,202][23] We have previously demonstrated correlative TEM and NanoSIMS imaging to assess the neurotransmitter content at the nanometer vesicles in cells. 13,14rotein turnover has been studied by other imaging techniques, especially uorescence microscopy. 9,10Fluorescence imaging provides protein turnover information based on organelle specic proteins (for example, using TOM20 or TOM70 to represent mitochondria), and thus can only determine the turnover of these proteins but not the whole organelles.In addition, dysfunctional organelles, which may not express specic proteins, could be le out from the analysis. 22ur work using correlative TEM/NanoSIMS provides imaging of a collection of many organelles within single cells based on their morphological properties.This allows a wide range of comparisons of protein turnover across different types of organelles to obtain an insight into the molecular and functional relations between them.
In this study, we employed correlative transmission electron microscopy (TEM) and NanoSIMS imaging to study the subcellular protein turnover in neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSC) using different isotopic amino acids as the protein precursors.Here, TEM imaging aided in the identication of specic subcellular structures, which are then correlated with the NanoSIMS images of the same cells to extract the corresponding turnover rates.We incubated NPCs for a determined period with one of the isotopic amino acids, 15 N-containing leucine, proline, glycine, alanine or phenylalanine, which were uptaken by the cells and incorporated into newly synthesized proteins (pulse period).We then chemically xed the cells at different time-points, following a 6 h to 96 h chase period aer the amino acid incubation.By correlating TEM and NanoSIMS imaging, we could localize the incorporated 15 N-containing amino acids in different organelles within single cells at various chase times.We found that protein turnover in different organelles was signicantly different, and that different isotopic amino acids showed different apparent levels of protein turnover within the cells.In this study, we used TEM and NanoSIMS imaging as a valuable tool to investigate the subcellular maps of chemical composition and protein turnover in human neural progenitor cells.This is applicable to various biological questions related to protein turnover, such as protein turnover during the cell differentiation, development and degradation, protein homeostasis in cell and organismal behaviours, protein turnover in aging and neurodegenerative diseases, and manipulating protein turnover for therapeutic applications.With this study, we introduce a groundwork for further investigation into the functional regulation of protein turnover in iPSC-derived cells at the single organelle level.

Neural progenitor cell (NPC) culture
Midbrain NPCs derived from human induced pluripotent stem cells (hiPSCs) were obtained from the Carl Ernst lab, McGill University, Montreal, Canada.The use of these human cells was approved by the Research Ethics Board of the Centre intégré universitaire de santé et de services sociaux de l'Ouest-de-l' Îlede-Montréal with the ethics approval code F9H-749 and the date of approval of May 13, 2022.NPCs were thawed rapidly at 37 °C aer which they were transferred to 10 ml of warm NPC medium (STEMDiff Neural Progenitor Basal Medium, Catalog #05834 STEMCELL technologies, Canada).The cells were centrifuged immediately at 1460 rpm for 5 min and the cell pellet was subsequently resuspended in NPC medium with 200 ng ml −1 Sonic Hedgehog (genescript Cat# Z03067); NPCs were plated onto a poly-D-lysine (Cat# P7280, Sigma-Aldrich, Sweden) and Laminin (Cat# L2020, Sigma-Aldrich, Sweden) coated T25 ask (Nunc™ EasYFlask™, Fisher Scientic, Sweden) and kept in an incubator at 37 °C and 5% CO 2 .Medium was exchanged every two days and the cells were propagated when they reached conuency.For protein turnover experiments, NPCs were plated on poly-D-lysine and laminin coated glass bottom dishes (Mat-Tek Life Sciences, Ashland, MA USA), and were incubated with 4 mM 15 N-containing amino acids for 48 h, followed by an incubation with regular NPC medium for a pre-determined chase period.Aerward, the cells were xed by warm 2.5% glutaraldehyde in 0.1 M PIPES buffer.

Sample preparation for correlative TEM and NanoSIMS
Samples were prepared for correlative TEM and NanoSIMS as previously described by Nguyen et al. 13 Monolayer of cells embedded in resin were sectioned at 150 nm thickness.The workow of sample preparation is presented in Fig. 1.

TEM imaging
The sample sections were placed on copper nder grids with carbon support lm (Electron Microscopy Sciences, FCF200F1-CU).TEM imaging was performed with a Thermo Scientic™ Talos L120C TEM microscope at the Centre for Cellular Imaging (Sahlgrenska Academy, University of Gothenburg, Sweden).The instrument was operated at 120 keV, and TEM images were taken at a magnication of 8500.

NanoSIMS imaging
NanoSIMS imaging was performed with a NanoSIMS 50L (Cameca, France).Before the measurement, the samples were sputter coated with a thin lm of gold to ensure good conductivity.A 16 keV Cs + primary ion beam was used for implantations and image acquisitions.The same cells which had been imaged with TEM were located and implanted with a uence of 3 × 10 16 Cs + per cm 2 .Image acquisition was performed with D1-4 (diaphragm width of 150 mm) and a primary ion beam current of 0.9-1 pA.Pixel size ranged from 45 to 82 nm with the FoV ranging from 23 to 42 mm.Dwell time was set at 5 ms per pixel.Each area was acquired with 5 consecutive image planes.
Detectors were tuned to measure 12

Image analysis
TEM and NanoSIMS images were overlayed using Affinity Designer, where regions of interest (ROIs) of selected organelles were drawn on the TEM image for each cell.Binary images outlining the ROIs and matching the same pixel size of the corresponding NanoSIMS image were created and opened in ImageJ.The ROIs were automatically detected with the LabelstoROI plugin to ImageJ. 24Subsequently, NanoSIMS images were opened in the OpenMIMS plugin to ImageJ.Consecutive image planes were dri-and dead-time corrected, accumulated, and a ratio HSI image of 12 C 15 N − / 12 C 14 N − was generated.Isotopic ratios of 12

Correlative TEM and NanoSIMS imaging as a tool for investigating subcellular turnover
To investigate subcellular protein turnover, a workow of sample preparation and correlative TEM and NanoSIMS imaging is presented in Fig. 1.NPCs were incubated with isotopic ( 15 N) amino acids for 48 h (Fig. 1A).This was followed by an incubation with regular cell medium for different periods allowing to track the uptake and subsequent clearance of the isotopic amino acid via newly synthesized proteins (Fig. 1B).Aerwards, the cells were chemically xed with glutaraldehyde, then treated with osmium tetroxide and uranyl acetate, and embedded into epoxy resin as a monolayer to keep the cellular structure intact (Fig. 1C).The samples were subsequently cut into 150 nm sections and placed onto copper nder grids (Fig. 1D).TEM imaging was performed prior to NanoSIMS imaging (Fig. 1E and F) due to the former being non-destructive and the latter eroding the sample surface during the measurement.Secondary electron images can also be obtained with the NanoSIMS in parallel with the measurement of negative ions, however they only provide the information of morphology of the sample surface, and thus cannot be used to observe subcellular structures such as organelles beneath the sample surface (Fig. S1 †).With the above-described workow, TEM and NanoSIMS images of the same cell were obtained (Fig. 1G) and the isotopic 15 N enrichment (d 15 N) of specic organelles within the cell was extracted by overlaying the images from these imaging modalities.
Isotopic enrichment (d 15 N) in per mille (&) was calculated as the ratio of 12 C 15 N − over 12 C 14 N − above the natural atmospheric ratio (r air = 3.7&) using the following eqn (1): where 15 N/ 14 N is detected as 12 C 15 N − / 12 C 14 N − .The Poisson uncertainty (eqn (2) in ESI †) of the measurements within the regions of interest (ROIs) typically ranged from 5-50& with the highest value of 120&.Given the relatively high d 15 N values measured in the cells (up to 22 323&), this is deemed as an acceptable uncertainty.For the smallest, least 15 N-enriched ROIs where the uncertainty is expected to be the highest, the uncertainty of each measurement was plotted in Fig. S2.† Protein turnover can be tracked via 15 N enrichment in the cells owing to the ability of NanoSIMS, given the proposed sample preparation, to mainly detect the bound 15 N-amino acid, which is mostly via incorporation into proteins. 25,26mall metabolites containing 15 N-amino acids are not sufficiently xed by glutaraldehyde and therefore will be washed away during multiple washing steps of the sample preparation.To support the argument that the 15 N enrichment is mainly attributed to proteins, not small metabolites, we treated NPCs with a cocktail of protein synthesis inhibitors (puromycin, cycloheximide, and geneticin) and with 15 N-labelled amino acid ( 15 N-leucine or 15 N-glycine).The results showed that the cells treated with protein synthesis inhibitors reduced the 15 N enrichment by over 90% compared to that in the cells without the inhibitor treatment (Fig. S3 †).This supports the claim that proteins are the major attribution to 15 N enrichment in the cells.
Using the same experimental procedure, we could also measure simultaneously other abundant bound isotopes, 31 P − and 32 S − .We rst looked at the isotopic distribution from endogenous molecules, 31 P − and 32 S − , across the single NPCs.Both 31 P − and 32 S − signals were pooled from all conditions and normalized to the respective 12 S1  and S2 †).Mitochondria and centrosomes show increased levels of 32 S − compared to other cellular compartments, which is possibly explained by a high protein density in these organelles compared to other compartments.It is possible that methionine and cysteine, amino acids containing sulphur, are present in a high abundance in these organelles leading to a high content of 32 S − .In addition, a high amount of iron sulphur clusters, which are involved in DNA metabolism and are needed for mediating electron transfer in mitochondria, can give rise to a high content of 32 S. 27,28 On the other hand, the highest level of 31 P − was observed in nucleoli, nucleus, and lamellar inclusions, most likely due to an elevated content of nucleic acids and phospholipids in these structures.

Protein turnover is heterogeneous at individual organelles in NPCs
We examined the subcellular protein turnover of NPCs via the spatial distribution of 15 N enrichment across the cell using precursor 15 N-glycine.It can be endogenously synthesized (e.g., from serine, 29,30 threonine 31 and choline 32 ) but usually not at a sufficient level.Moreover, glycine is also a neurotransmitter which, upon binding to its receptor, allows chlorine ions to diffuse across the membrane. 33ight different cellular compartments were identied on the TEM images, i.e., nucleus, nucleolus, mitochondria, endoplasmic reticulum (ER), Golgi, vacuoles, lamellar inclusions, and vesicles (Fig. 2C, D, G, H, K, L, O and P, respectively), and Edge Article Chemical Science their 15 N enrichment was measured from the overlaid Nano-SIMS images of the same cells.The 15 N enrichment of these compartments was examined at ve chase time-points, from 6 to 96 h (Fig. 2).We found that the 15 N enrichment is heterogenous across the organelles of individual NPCs.The cells xed aer the 6 h chase time following amino acid incubation exhibit the highest 15 N enrichments, which then decrease at the later chase times following an exponential decay trend.This is consistent with other previous studies on the decay t of protein turnover. 9 13C 6 -lysine amino acid from food has been shown to rapidly equilibrate (within 24 hours) as the main source of intake for protein synthesis in mouse brain using a mathematical model and SILAC experiments. 6Therefore, with an isotopic incubation period of 48 h used in our study, it is expected that the 15 N signal of the protein turnover will drop almost immediately from 0 h when the isotopic incubation is nished.At the 6 h time-point, the nuclei, nucleoli and mitochondria display the highest 15  With increasing chase periods, all organelles of NPCs showed a decrease in 15 N enrichment, although at different rates, indicating that the 15 Nlabelled proteins of particular organelles are replaced gradually by newly synthesized unlabelled proteins.The high turnover of nucleoli can be related to their function in ribosome biogenesis, cell cycle regulation and gene expression. 34he high turnover rate of mitochondria could be explained by a high energy demand in NPCs, as mitochondria generate energy through the Krebs cycle, but also by the ability of mitochondria to mediate cell growth. 35Previous studies in in vivo mouse brains and neurons showed that a majority of mitochondrial proteins have relatively low turnover due to their long lifetime (>10 days). 3A few short-lived mitochondrial proteins have faster turnover and tend to be involved in fatty acid metabolism and peroxisome function.In our study, mitochondria showed a high rate of turnover over 96 h chase period (K = 0.01116), which is among the highest turnovered organelles such as nucleoli and vacuoles.The difference between our nding and the previous study could be due to the immature state of our NPCs and differences in protein lifetimes in vitro compared to in vivo.Recent evidence showed that mitochondria play an important role in NPCs in regulating neurogenesis processes and the metabolic programming required for cell fate decision. 36These regulating functions together with the classical role of mitochondria as an energy generator for cellular homeostasis could lead to their highly active function in NPCs.How actively the cellular compartments function, in turn, has been shown to closely relate to protein turnover, particularly a strong connection was found between single synaptic activity and protein turnover. 10Therefore, it is speculated that the high rate of protein turnover of mitochondria in NPCs is a result of an unusual high activity of the organelles for regulating cell fate and neurogenesis.
In contrast to mitochondria, vesicles exhibited the lowest 15 N enrichment level and the lowest rate of protein turnover.
Vesicles were shown to release around 200 times before being inactivated, 15 and immature NPCs are expected to undergo far fewer neuronal secretion events compared to mature neurons, indicating a low need of protein synthesis and turnover of vesicular proteins.
It is noted that the level of 12 C 14 N − was shown signicantly different across organelles.For example, a high abundance of 12 C 14 N − was found in mitochondria and nucleoli, whereas vesicles exhibited the lowest amount (Fig. S5 and Table S3 †). 12 C 14 N − is a component of many biomolecules within cells such as nucleosides, phospholipids, and proteins.The nding here is consistent with the data obtained by Lange et al. who reported a heterogenous distribution of 12 C 14 N − , as well as 32 S − and 31 P − , across different organelles in HeLa cells. 22To test whether high 15 N enrichment is due to high 12 C 14 N − content of the respective organelle, the relationship between 12 C 15 N − enrichment and 12 C 14 N − levels were illustrated in Fig. S6.† However, no consistent correlation between 15 N enrichment and 12 C 14 N − levels was observed when looking at all the ROIs of each amino acid in each time-point.In addition, any correlation between 15 N enrichment and 12 C 14 N − levels would be negligible when comparing 15 N enrichment over time (turnover rate) between different organelles, therefore the differences in the protein turnover rate are due to the functional roles of the organelles and are not mere differences in the content of 12 C 14 N − .Despite being diluted by cell mitosis over the 96 h chase period, the level of 15 N enrichment of all cells is still signicantly above the natural abundance of 3.7& and the control cells (∼−38&) which were incubated in regular cell medium without 15 N-glycine (Fig. S7 †).The dilution due to cell mitosis does not appear to be the dominating factor for the decrease in 15 N enrichment as conrmed by an experiment with NPCderived postmitotic cells, which were cultured with 15 N-glycine in a differentiation cell medium inhibiting cell mitosis.The result showed a decreasing trend of 15 N enrichment over a 96 h chase period similar to that obtained with the NPCs (Fig. S8 †).This indicates that the decrease in 15 N enrichment in the cells is mainly driven by the turnover of the cellular proteins, and not by cell division.The d 15 N level aer 96 h is also signicantly above the d 15 N in the resin surrounding the cells.The resin adjacent to the cells shows higher enrichment compared to that of resin areas further away from the cells, possibly due to the extracellular matrix components containing a certain 15 Nlabelled proteins having been secreted from the cells (Fig. S9 †).The remaining elevated level of 15 N over 96 h indicates a considerable proportion of proteins living longer than 96 h in the cells.In addition, it could simply be due to other metabolic pathways of 15 N-glycine and its biosynthesis (e.g., into serine), 37 which further circulate 15 N within the cellular system.

Different protein precursors result in specic subcellular turnover patterns in NPCs
Different amino acids are transported into the cell via uniporters, symporters and antiporters; for example glycine can be transported via symporter SNAT requiring Na + as a cofactor whereas leucine is up-taken via antiporter LAT1. 38Some amino acids such as phenylalanine and leucine are termed "essential" as they cannot be synthesized by the body and therefore need to be supplied exogenously.The different amino acids participate in different functions of a protein due to their different charges, polarity, or hydrophobicity.In addition, amino acids can be transformed into other amino acids, 37 neurotransmitters (e.g., tyrosine, phenylalanine) 39 and other metabolites. 40Therefore, different amino acids are likely to contribute differently to the overall protein homeostasis within the cells.We compared the subcellular protein turnover from ve 15 N containing amino acids, which differ in the size of the side chain, and being aliphatic or aromatic.Similar to the previous section, eight different cellular compartments were identied (Fig. 3A-D) and 15 N enrichment of those was relatively quantied using correlative TEM and NanoSIMS imaging.We observed that the incorporation of these amino acids into the cells is largely different.NPCs incubated with 15 N-leucine, one of the most frequently encoded amino acids, display the highest 15 N enrichment, ranging up to 2.2 × 10 4 &, compared to the other amino acids in all organelles, in a range of d 15 N ∼2 × 10 3 -1 × 10 4 & (Fig. 3E-L).The lowest was found with 15 N-phenylalanine which is incorporated into the nucleus and nucleoli at d 15 N ∼2.5 × 10 3 &.In addition, the enrichment in specic organelles is varied for particular isotopic amino acids.Interestingly, mitochondria were found to have the highest 15 N abundance, whereas the lowest amount of 15 N was found in vesicles, followed by lamellar inclusions, for all the ve 15 N containing amino acids.Golgi showed the highest 15 N level in the cells incubated with 15 N-leucine, and 15 N-phenylalanine.On the other hand, nuclei and nucleoli showed the highest 15 N level with 15 N-glycine, but the lowest with 15 N-phenylalanine.ER and vacuoles mostly exhibited a mid-range 15 N enrichment.
There is a variation in the 15 N enrichment within the analysed cells, especially the variation for 15 N-leucine incubation is signicantly larger than those for other amino acids, noticeably at the rst and second chase time points (Fig. 3E-L, and S10 †).The variation is possibly caused by the varied phases of the cell cycle in NPCs, e.g., S-phase (DNA synthesis) or G1/G2-phase (cell growth), which could require different levels of protein synthesis and metabolism related to 15 N-amino acids, hence leading to a biological variation among cells.Leucine showed the largest variation which could be due to its initial enrichment level being much higher than those of the others, thus a larger scale of variation is observed.
The clearing of 15 N within these organelles was also different over the chase period of 6-96 h (Fig. 3E-L).The one-phase decay ts of the turnover across organelles for each 15 N-amino acid are presented in Fig. S11-14.† Across all the examined organelles, 15 N-leucine was enriched the most but did not exhibit the highest turnover, which was observed with 15 N-proline (for example, the protein half-life t 1/2 ∼ 59 h in mitochondria for leucine but t 1/2 ∼ 42 h for proline, Fig. S11 and S12 †).Interestingly, 15 N-phenylalanine showed a signicantly lower turnover rate compared to the other amino acids.The 15 N enrichment detected in all organelles over 96 h chase time for 15 N-phenylalanine was only slightly lower than the initial enrichment.On the other hand, 15 N-alanine displayed the second highest 15 N enrichment in all the organelles, aer 15 Nleucine, and followed by 15 N-glycine and 15 N-proline.The latter two were at an approximately similar enrichment level in most compartments, except for nucleoli and mitochondria.The turnover of these amino acids in different compartments, however, followed different rates.Among the ve examined 15 N containing amino acids, leucine (containing a large aliphatic chain) and phenylalanine (with an aromatic large side chain) show the largest signicant difference in the 15 N incorporation, with the aliphatic one showing the highest enrichment and the aromatic one showing the lowest.The other smaller aliphatic chained 15 N precursors have an overall enrichment below that of aliphatic large-chained leucine.
Most of the amino acids oen undergo various metabolic pathways besides being precursors for protein synthesis.Leucine has been known to promote protein synthesis via an activation of the mammalian target of rapamycin (mTOR) pathway, and to be involved in energy production by converting to a-ketoisocaproate (a-KIC) and b-hydroxy-b-methylbutyrate (HMB). 41The role of leucine in stimulating protein synthesis was studied in muscles of neonatal pigs, 42 rats, 43 and humans. 44,45These metabolic functions, especially the stimulation of protein synthesis, could be the cause for the high initial level of enrichment of 15 N-leucine compared to the other precursors.
In contrast to leucine, 15 N-phenylalanine presented the lowest 15 N incorporation as well as the lowest turnover.This could be due to free 15 N-phenylalanine being oxidized generating m-tyrosine, which can be incorporated into proteins and result in cellular stress. 46Cellular stress in turn could inhibit the overall protein turnover, 16 resulting in a atter curve of the turnover of 15 N-phenylalanine across all the cell compartments.NPCs incubated with 15 N-phenylalanine show high enrichment in mitochondria aer 6 h chase time.
For all the examined 15 N-containing amino acids, the 15 N enrichment in the cells does not return to the natural abundance, or to the levels seen in the resin surrounding the cell, over 96 h chase period except for proline.It is possible that the 15 N-amino acids are metabolized into other molecules, e.g., the derived metabolites which remained in the cell for longer time or other amino acids being incorporated back into proteins.Another possibility is that a proportion of proteins living longer than 96 h remain in the cells.This elevation of 15 N enrichment aer 96 h was not pronounced in the cells incubated with 15 Nproline.Proline metabolism is the main pathway for generating glutamate and glutamine via a conversion into pyrroline-5carboxylate (P5C) under the catalysis of proline dehydrogenase. 47Glutamate and glutamine play both roles as amino acid and neurotransmitters in the brain and are involved in multiple metabolic pathways. 48,49Glutamate itself is the most abundant free amino acid and the major excitatory neurotransmitter in mammalian brain. 50These extended metabolic and signalling pathways could explain the low level of 15 N enrichment aer 96 h, therefore also a high rate of turnover of proline compared to the other precursors having similar initial enrichment as observed in our data.N-leucine (dark purple) 15 N-alanine (green), 15 N-glycine (black), 15 N-proline (light purple), and 15 N-phenylalanine (pink) within different organelles.NanoSIMS measurements were obtained after 15 N-amino acid incubation and a clearing period of 6, 12, 24, 48, 96 h.A one-phase decay curve was fitted to each dataset with GraphPad Prism 9.3.1 software.
It is noted that NanoSIMS measurement only detects small, fragmented ions, therefore the identity of the molecules associated with the 15 N label cannot be identied.However, our data indicates that different protein precursors undergo specic protein synthesis and cellular metabolic pathways which result in distinguished subcellular patterns of protein turnover across the cell.Using NanoSIMS, it is possible to image and compare the differences in the protein turnover across different types of organelles, and even between the same organelles, within single cells (Fig. S15 †), which has not been possible with pool analysis

Molecular lifetime is inuenced by subcellular localization and protein precursors
We determined the half-lives of total proteins and molecules associated with 15 N containing amino acids across different cellular compartments.The half-lives were found to be varied not only by compartments but also by 15 N-amino acids (Fig. 4).First, NPCs incubated with 15 N-proline showed the shortest half-lives in most of the organelles (in a range of 29-45 h), except for the lamellar inclusions (68 h), followed by 15 N-leucine, 15 N-alanine, 15 N-glycine, and 15 N-phenylalanine.This is as expected for 15 Nproline due to its extensive metabolic and signalling pathways leading to a high rate of turnover, as explained earlier.Second, we observed that 15 N-alanine has rather similar half-lives in all the organelles (within 10 h variation) with no statistically signicant differences.This suggests that the subcellular turnover of 15 Nalanine is homogenous and that its metabolism is rather consistent across organelles.Third, NPCs incubated with 15 Nphenylalanine show extremely long half-lives of around 200 h or more.This could be due to cellular stress, as explained earlier, affecting cellular metabolism and turnover leading to a very slow clearance of the isotope.In turn, this affects the t of the curve and the half-life calculation.
Protein half-lives were found signicantly different across cellular organelles under different 15 N-containing amino acids incubations.In Fig. 4B, the half-lives show the largest signicant differences between the examined organelles following 15 N-leucine incubation, except for lamellar inclusions due to their high variation. 15N-glycine and 15 N-proline exhibit differences between a few organelles, such as mitochondria, nucleus and vacuoles, but at lower signicance levels than 15 N-leucine.Considering the protein half-lives within individual organelles from the four 15 N-amino acids ( 15 N-phenylalanine was excluded due to its cell stress effects, and thus the decay trend does not t) (Fig. S16 †), mitochondria show the largest difference between these precursors followed by ER, nucleus, and vacuoles.Interestingly, 15 N-glycine and 15 N-leucine result in rather similar protein half-lives in mitochondria, ER, and vacuoles, but signicantly different ones in nucleus and nucleolus.On the other hand, vesicles and lamellar inclusions exhibit similar protein half-lives from all these precursors.
The data indicates that protein lifetime is inuenced by subcellular localization, which is likely related to the biological functions of proteins within specic cellular structures.This is supported by both the in vivo, and in vitro data of other studies (Fig. S17 †).Furthermore, the protein lifetime is also linked to the incorporating amino acids, which not only play as protein precursors but also as mediators in the crosswalks of multiple cellular metabolic pathways.

Conclusions
We employed correlative TEM and NanoSIMS imaging to study subcellular protein turnover of human NPCs.The incorporation of isotopically labelled amino acids, as protein precursors, the protein turnover rates and the chemical composition were found signicantly heterogenous at the organelle level.In addition, subcellular turnover patterns were distinguished between different precursors, possibly owing to their involvement in other cellular metabolic pathways.Furthermore, general protein lifetime was found to be inuenced by subcellular localizations of proteins and by specic protein precursors.The novelty of our work is summarised in three main aspects.First, the nding about the heterogenous cellular protein turnover at the organelle level indicates a relation between protein turnover and subcellular activity.This possibly introduces a new paradigm for further discoveries in modulation of organelle turnover to inuence cellular activity and processes.Second, the nding about the distinct subcellular turnover patterns for different amino acids provides a new perspective of subcellular protein turnover.This is particularly a very useful information in the eld of protein turnover using SILAC method, where most studies use a single amino acid (primarily lysine) as a protein precursor, and thus could have a lysine bias.Third, the application of NanoSIMS to study protein turnover in human neural stem cells has never been done before at the subcellular level.Previous studies mostly focused on organelle specic proteins, but not a collection of organelles within single cells.This could be a groundwork for further studies in human stem cell biology to understand the subcellular regulation driving stem cell differentiation and stem cell fate.
We have demonstrated correlative TEM and NanoSIMS as a powerful integrative imaging tool to obtain complementary properties and composition of samples to investigate complex cellular processes such as molecular turnover.Applications of this method in highly polarized cells such as neurons and using detailed pulse-chase experiments with other metabolites to test the turnover of specic metabolic pathways will provide better insight into neuronal processes and brain biology.In this work, we compared the differences in turnover between different amino acids for each type of organelles in different groups of cells, meaning that each group of cells were incubated with one particular isotopically labelled amino acid.However, it would also be interesting to examine the turnover of multiple amino acids in the same cells for which labelling with multiple isotopic amino acids (e.g. 13 C − , 15 N − and 18 O − ) is needed.This can be considered for the future work.

Fig. 1
Fig. 1 Schematic of sample preparation and correlative TEM and NanoSIMS imaging.(A) Samples are incubated with 15 N-containing amino acid.(B) Cell medium with the labelled amino acid is replaced with regular cell medium and incubated for different time periods (chase time).(C) After the respective times, cells are fixed with Glutaraldehyde (GA), OsO 4 , and Uranyl Acetate (UA) and subsequently dehydrated and embedded in epoxy resin.(D) The resin block containing a monolayer of cells is then sectioned into 150 nm thick sections, which are placed on copper grids coated with Formvar.(E) Samples are imaged by TEM first, to obtain the ultrastructure of the cells.(F) The cell areas that were imaged with TEM are then imaged with NanoSIMS, to obtain their chemical composition.(G) Example NanoSIMS and TEM images of an NPC.TEM image, NanoSIMS ion images of 12 C 12 C − , 14 N 12 C − , 15 N 12 C − , a hue saturation intensity (HSI) ratio image ( 15 N 12 C − / 14 N 12 C − ), and an overlay of TEM and HSI ratio images.NPCs were incubated with 15 N-glycine for 48 h and underwent a chase period of 12 h.Arrows indicate different organelles which can be identified as mitochondria (blue), nucleolus (red), and lamellar inclusion (yellow).The colour scale represents the 15 N 12 C − / 14 N 12 C − ratio with blue at 0.0037 (natural abundance) and magenta at 0.04 (d 15 N ∼ 1 × 10 4 &).Scale bar 10 mm.
C 15 N − / 12 C 14 N − for designated ROIs were extracted, calculated into isotopic enrichment (d 15 N) values (per mille, &), and then plotted with the soware GraphPad Prism 9.3.1.

C 2 −
signal (Fig. S4 †).Across most of the identied organelles, the signals of 32 S − and 31 P − were shown to be signicantly different based on the one-way ANOVA tests (Kruskal Wallis, Dunn's multiple comparison) (Tables

Fig. 2
Fig. 2 Protein turnover is different across different cellular compartments of single NPCs.NPCs were incubated with 15 N-glycine for 48 h, and then with regular cell medium for different time periods (chase time: 6, 12, 24, 48, 96 h).The 15 N turnover rate is plotted for each organelle and example correlative TEM and NanoSIMS images of the corresponding organelle are shown.(A&C) Nuclei, scale bar 5 mm in C; (B&D) Nucleoli, scale bar 1 mm in D. (E&G) Mitochondria, scale bar 1 mm in G; (F&H) ER areas, scale bar 500 nm in H. (I&K) Golgi, scale bar 500 nm in K; (J&L) vacuoles, scale bar 500 nm in L. (M&O) lamellar inclusions, scale bar 500 nm; (N&P) vesicles, scale bar 500 nm in P. A one-phase decay curve is fitted to each graph using GraphPad Prism software (constraints: Y0 > 0, plateau = 0, K > 0); R 2 coefficient of determination, K rate constant, and t 1/2 half-life.Example images are from cells incubated with 15 N-glycine and 12 h chase time.Cellular compartments are indicated with a yellow dashed line.The colour scale (bottom right) represents the 15 N 12 C − / 14 N 12 C − ratio ranging from the natural abundance (0.0037, blue) to d 15 N ∼1 × 10 4 & (0.04, magenta) for all images.

Fig. 3
Fig. 3 Different amino acids show distinguished protein turnover patterns at subcellular level.(A & C) Representative TEM images of (A) nucleoli and (C) mitochondria.(B & D) NanoSIMS ratio HSI image 15 N 12 C − / 14 N 12 C − for nucleoli and mitochondria with 12 h chase time after the incubation with 15 N-leucine, 15 N-alanine, 15 N-glycine, 15 N-proline, and 15 N-phenylalanine (left to right).Cellular compartments are indicated with a dashed line.The colour scale (below D) represents the 15 N 12 C − / 14 N 12 C − ratio ranging from natural abundance level (0.0037, blue) to d 15 N ∼1.2 × 10 4 & (0.05, magenta) for all NanoSIMS images.Scale bars are 500 nm.(E-L) 15 N turnover rate plotted for the incubation with 5 different amino acids:

Fig. 4
Fig. 4 Protein lifetime within individual organelles.(A) NanoSIMS HSI ratio images of 15 N 12 C − / 14 N 12 C − for different organelles with different chase time of 6, 12, 24, 48, 96 h after 15 N-glycine incubation.Cellular compartments (indicated with a dashed line) are identified by correlating with TEM images of the same cell areas.The colour scale (bottom) represents the 15 N 12 C − / 14 N 12 C − ratio ranging from natural abundance level (0.0037, blue) to d 15 N ∼1.2 × 10 4 & (0.04, magenta) for all images.Scale bars for nuclei: 5 mm, for nucleoli 1 mm and 500 nm for all other organelles.(B) Protein half-lives (t 1/2 ) within different subcellular compartments after incubation with different 15 N-amino acids.SEMs for t 1/2 were calculated from 95% confidence interval of the t 1/2 of the one-phase decay fits.One-way ANOVA (Brown Forsythe and Welch) and subsequent Dunnett's multiple comparison test were performed, and significances are indicated above (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).